Ubiquitin, a 76 residue polypeptide is used as a posttranslational modification to alter intracellular protein functions in eukaryotic cells. Historically, the ubiquitylation system was identified as an ATP-dependent signal for targeting intracellular proteins for proteasomal degradation (Hershko, A. & Ciechanover, A., 1998, Ann. Rev. Biochem. 67, 425-479; Wilkinson, K. D., 2000, Sem in Cell & Dev. Bio., 11, 141-148 and Varshaysky, A., 2012, Ann. Rev. Biochem, 81, 167-176)).
Ubiquitylation of proteins is a multi-step process requiring the sequential action of three enzymes: ubiquitin-activating enzymes (E1s) activate ubiquitin that is subsequently loaded onto ubiquitin-conjugating enzymes (E2s) and finally, the ubiquitin is covalently linked to a lysine side-chain from the E2s via specific recruitment of the target protein, and facilitation of the transfer by ubiquitin ligases (E3s). Ubiquitin can be linked to target proteins singly, to form monoubiquitin adducts, however, in many cases, the initial ubiquitin is then extended by the covalent attachment (again by E1, E2 and E3 proteins) of additional ubiquitin moieties to form poly-ubiquitin chains. Moreover, as any one of ubiquitin's seven internal lysine residues or its amino terminus can serve as sites for conjugation, the resulting poly-ubiquitin chains can have various, highly distinct topologies with different biochemical and biological functions. While Lys-48 (K48)-linked poly-ubiquitylation of proteins is widely recognised as a critical pathway for protein degradation, many additional roles have been attributed to either poly-ubiquitylation of proteins via non-K48 chains, linear ubiquitin chains as well as mono-ubiquitylation of proteins (Hicke, L., 2001, Nature Reviews Mol Cell Bio, 2, 195-201; Ikeda, F. & Dikic, I., 2008, EMBO Reports, 9, 536-542; Iwai, K., 2012, Trends in Cell Biology, 22, 355-364 and Komander, D. & Rape, M., 2012, Ann. Rev. Biochem, 81, 203-229). In addition to post-translational modification by ubiquitin, a whole family of ubiquitin-like (Ubl) modifications have been described. The degree of conservation between ubiquitin and ubiquitin like factors is somewhat limited at the protein sequence level; however, all members of the family share similar overall three-dimensional structures and highly related mechanisms of conjugation to their respective targets involving E1, E2 and E3 enzymes (Hay, R. T., 2007, Trends in Cell Biology, 17, 370-376; Hochstrasser, M., 2009, Nature 2009 (458) 422-499 and van der Veen, A. G., & Ploegh, H. L., 2012, Ann. Rev. Biochem, 81, 323-357).
Furthermore, since conjugation with ubiquitin or ubiquitin like molecules is a crucial post-translational modification that regulates cellular processes in eukaryotes, it is a system that pathogens encounter when attempting to infect humans and animals. Modification with ubiquitin or Ubl plays a central role in defence systems, for example. Thus, pathogens such as bacteria, viruses, fungi and parasites have evolved to exploit or evade the host systems for their own benefit, in order to maximise their chances of establishing a successful infection (Calistri et al, 2014, Cells, 386-417).
As for other protein post-translational modifications, conjugation of ubiquitin or ubiquitin-like factors to target protein is reversible, this being mediated by isopeptidase enzymes that are often collectively referred to as deubiquitylating enzymes or DUBs. DUBs comprise a large class of intra-cellular peptidases that cleave ubiquitin from polypeptide substrates.
Their substrates can be ubiquitin precursors, ubiquitin adducts, poly-ubiquitin chains, monoubiquitylated proteins or poly-mono-ubiquitylated proteins (Iwai, K., 2012, supra). If ubiquitin-like peptidases are included, over a hundred DUBs are encoded by the human genome. DUBs can be classified into five families: ubiquitin carboxyl-terminal hydrolases (UCH), ubiquitin specific proteases (USPs), ovarian tumour proteases (OTU), MJD (Josephine) and MPN+/JAMM (JAB1/MPN/MOV34 metallo-enzymes). The first four families are cysteine peptidases, while MPN+/JAMMs are metallopeptidases (Reyes-Turcu, F. E., et al, 2009, Ann. Rev. Biochem, 78, 363-397 and Sacco J. J., et al, 2010, IUBMB Life, 62, 140-157). In addition to processing ubiquitin and ubiquitin adducts, some USPs have been shown to selectively process specific ubiquitin-like proteins (for example, USP18 acts on the ubiquitin-like protein ISG15) (Zhang, D. & Zhang, D. E., 2011, J. Interferon and Cytokine Research, 31, 119-130)). In the case of the SUMO family of ubiquitin-like proteins, adducts are reversed by a specialised group of DUBs termed SENPs, all of which are cysteine peptidases, and some of which may also remove NEDD8. (Hay, 2007, supra and Dou, H., et al, 2010, Molecular Cell, 39, 333-345).
Similarly, microorganisms which have the ability to infect eukaryotic organisms (pathogens) have developed enzymes to reverse the conjugation of ubiquitin and Ubl molecules to their target protein, or have evolved strategies to affect the host enzymes. DUBs have been described for microorganisms.
While all DUBs are peptidases, there are considerable differences between their precise mechanisms of action, and there are also major differences in the regulatory mechanisms that modulate DUB selectivity and specificity (Komander, D., et al, 2009, Nature Reviews Mol Cell Bio, 10, 550-563). In this regard, DUBs can be classified into three main categories according to their type of substrate cleavage activity: some generate free ubiquitin from linear substrates, such as poly-ubiquitin chains or ribosomal protein fusions; others liberate ubiquitin from proteins modified post-translationally on lysine residues; while, a third class comprises DUBs that edit poly-ubiquitin chains (Komander et al, 2009). For in depth discussions of DUB mechanism-of-action, we refer the reader to several excellent reviews on this subject (Reyes-Turcu, F. E., et al, 2009, Ann. Rev. Biochem, 78, 363-397; Linder, H. A., 2007, Virology, 362, 245-256; Sun, S. C., 2008, Nature Reviews Immunology, 8(7), 501-511; Hussaun, S. et al, 2009, Cell Cycle, 8, 1688-1697 and Ramakrishna, S. et al, 2011, Cell and Mol Life Sci, 68, 15-26).
Deubiquitylating enzymes may also be called deubiquitinating enzymes, deubiquitinating peptidases, deubiquitinases, ubiquitin isopeptidases, ubiquitin proteases, ubiquitin hydrolases, or DUBs.
The present invention relates to uses, methods and assays involving isopeptidase enzymes. An isopeptidase is an enzyme that catalyses the cleavage of an isopeptide bond, especially that between the terminal diglycine attached to ubiquitin, as well as cleavage of ubiquitin fusion or precursors through peptide bonds. As discussed above, deubiquitylating enzymes are isopeptidases. Other isopeptidases include SUMO (Small Ub modifier) peptidases, ATG8 (Autophagy-related protein 8) peptidase, ISG15 (Interferon-stimulated gene 15) peptidase, NEDD8 (Neural precursor cell, developmentally down regulated 8) peptidase as well as any enzyme-cleaving adducts. Each isopeptidase catalyses the cleavage of an isopeptide bond involving a particular Ubl or Ub, and will be specific for the type of reaction it catalyses.
In order to monitor the activity of isopeptidases, particularly deubiquitylating enzymes, a number of tools and assays have been developed. A number of in vitro assays have been designed to characterise both deubiquitylating enzymes and inhibitors: the list includes many covalent adducts to the carboxy-terminus of ubiquitin (Layfield, R. et al, 1999, Anal Biochem., October 1999, 274(1): 40-49; Lee, J. I., et al, 1998, Biol Proced Online, July 20; 1; 92-99; Liu, C. C. et al, 1989 Dec. 5, J Biol Chem, 264(34): 20331-8; Falquet, L., et al, 1995, FEBS Lett, February 6; 359(1) 73-77; Larsen, C. N. et al, 1998, Biochemistry, March 10; 37(10): 3358-68; Dang L. C., et al, 1998, Feb. 17, Biochemistry, 37(7):1868-79 and Tirat, A., et al, 2005, Anal Biochem, 343(2): 244-55). These assays have led to the biochemical and biophysical characterisation of a large number of DUBs.
A number of ubiquitin-based activity probe assays targeting the catalytic sites of deubiquitylating enzymes in cell extracts have been published in the last decade (Borodovsky, A., et al, 2001, EMBO J., 20(18):5187-96; Borodovsky, A. et al, 2002, Chem Biol, 9(10); 1149-59; Ovaa, H. et al, 2004, PNAS USA, 101(8):2253-8; Hemelarr, J. et al, 2004, J. Proteome Res., 3(2):268-76; Ovaa, H. et al, 2005, Methods Enzymol., 399: 468-78; Galardy, P. et al, 2005, Methods Enzymol., 399:120-31, de Jong, A., et al, 2012, Chembiochem., 13(15):2251-8 and Love K. R., et al, 2009, ACS Chem Biol, 4(4):275-87). Ubiquitin-based activity probe assays have been successfully used to characterise deubiquitylating enzyme inhibitors (Altun, M. et al, 2011, Chem Biol, 18(11):1401-12 and Reverdy, C., et al, 2012, Chem Biol, 19(4): 467-77). Similar activity-based probe assays have also been developed for the characterisation of the activity of ubiquitin-like peptidases as well as non-human ubiquitin-like peptidases (An, H. & Statsyuk, A. V., 2013, J Am Chem Soc, 135(45):16948-62 and Claessen, J. H. et al, 2013, Chembiochem, 14(3):343-52). However, such probe assays are limited since they are low throughput, and as such are time and labour intensive.
Furthermore, a number of high throughput assays have been developed to monitor deubiquitylating enzyme activity in vitro. Many assays currently in use rely on cleavage of linear ubiquitin-fusions, (tetra-Ub, Ub-CEP52, Ub-GSTP1, Ub-DHFR, Ub-PESTc, etc.) that are synthesized recombinantly or chemically (Lee, J I, 1998, Larsen, C. N. 1998 and Baker, R. T et al, 1994, J Biol Chem, 269(41):25381-6). For small scale analysis of deubiquitylating enzyme activity, reaction products are analysed by gel electrophoresis, or are selectively precipitated and analysed by liquid scintillation spectrometry. Gel-based procedures are labour intensive and expensive, and while scintillation counting approaches are quantitative and allow processing of larger numbers of samples compared to gel-based assays, they require centrifugation and recovery of the supernatant. For higher throughput assays, fluorogenic substrates such as Ubiquitin-AMC (Ub-7-amino-4-methylcoumarin) or Ubiquitin-Rhodamine, have been employed, as well as the tetrapeptide z-LRGG-AMC, corresponding to the carboxyl terminus of ubiquitin (Dang, L C, 1998, Supra). Fluorescence Resonance Energy Transfer (FRET) has also been developed for high throughput deubiquitylating enzyme assays (Horton, R. A., et al, 2007, Anal Biochem, 360(1): 138-43). Bioluminescent and fluorescent quenching assays for deubiquitylating enzymes have also been recently developed (Orcutt S. J, et al, 2012, Biochim Biophys Acta., 1823(11): 2079-86 and Tian, X., 2011, 9(2): 165-73). The assays as described use AMC and FRET as detection means, however both assays suffer from the need for specialized custom reagents and equipment, as well as difficulty in adapting to a multi-well plate format from which the endpoints can be read directly. Such assays are used in an in vitro setting, and are biochemical. They do not measure the binding of an activity probe to an enzyme. Whilst these techniques may be suitable for high throughput biochemical assays in the in vitro setting they cannot measure the activity of isopeptidases in biological samples directly. These assays rely on reactions where the enzyme catalyses cleavage of a substrate mimic.
However, high throughput assays enabling the quantification of isopeptidase activity, particularly deubiquitylating enzyme activity in samples of cells or tissues are highly desirable. Such assays are highly advantageous, since they can provide information on the potential inhibition of the enzyme by an inhibitor, can monitor the cellular selectivity of a DUB, can monitor the pharmaco-dynamic activities of one or more enzymes and can be used as a diagnostic and prognostic tool. As such the assays can be used for determining and monitoring the development of one or more enzyme inhibitors, an important step in the drug discovery pathway. None of the currently available methods meets these requirements, and this has hindered or indeed prevented development of drugs which target isopeptidases, particularly deubiquitylating enzymes. The value of using biological samples comprising cellular materials, such as cells, tissues and biopsies is that a more robust picture of how the inhibitor in particular is working in situ and offers a wealth of information when compared to work on isolated enzymes.
The present inventors have recognised this unmet need in relation to the development of drugs that target isopeptidases, particularly deubiquitylating enzymes, and assays that provide diagnostic and prognostic information on isopeptidase activity, particularly deubiquitylating enzyme, activity. They have, therefore, developed a high throughput cell or tissue based assay that allows determination of the activity of isopeptidases. This determination can be performed for normal or pathological cellular material (biological sample). Such an assay has application in determining target engagement by putative inhibitors, monitoring pharmaco-dynamic activities of isopeptidases, and performing diagnostic and prognostic assays on patient-derived biopsies/biological samples.